In the yeast Saccharomyces cerevisiae, membrane phospholipids are synthesized by complementary (CDP-diacylglycerol and Kennedy) pathways, and the genes and enzymes in these pathways are regulated by genetic and biochemical mechanisms. The major hypotheses of the work proposed in this application are that phospholipid synthesis is regulated by phosphorylation and by mRNA stability. We will focus on the phosphorylation of choline kinase and the transcription factor Opi1p. Choline kinase catalyzes the committed step in phosphatdylcholine synthesis via the CDP-choline branch of the Kennedy pathway. Understanding its regulation is emphasized by the fact that unregulated levels of choline kinase activity is a common property of various cancers in humans. Opi1p is a negative transcription factor that controls expression of several phospholipid biosynthetic genes in response to inositol supplementation. Choline kinase is phosphorylated by protein kinase A, and we will examine the hypothesis that the enzyme is also phosphorylated by protein kinase C. Mutants defective in protein kinase C phosphorylation will be used to examine the consequences of protein kinase C phosphorylation on choline kinase activity, and on the regulation of phosphatidylcholine synthesis. Hierarchical phosphorylation of the protein kinase A and protein kinase C sites will be examined. Opi1p is phosphorylated by protein kinases A and C, and we will examine the hypothesis that Opi1p is also phosphoylated by casein kinase II. Mutants defective in phosphorylation will be constructed and used to examine the role of casein kinase II phosphoylation on Opi1p repressor function. Target site mutants for protein kinases A and C will be included in this analysis. Hierarchical phosphorylation of the protein kinase A, protein kinase C, and casein kinase II will be examined. mRNA stability is a mechanism by which the CDP-diaclyglycerol pathway is activated when the Kennedy pathway is blocked. Using mutants defective in phospholipid metabolism, we will examine the hypothesis that the signal for regulation of the CDP-diacylglycerol pathway by mRNA stability is a Kennedy pathway end product, a water-soluble intermediate of the Kennedy pathway, or a molecule derived from the turnover of a phospholipid synthesized via the Kennedy pathway. The mechanism for CHO1 mRNA (a CDP-diacylgylcerol pathway transcript) degradation will be examined.